Daniel:Notebook/ComboLock/2016-8-18
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Positive Control Amplicon Test
Buffer Prep
Wash/Binding Buffer
Reagent | Stock | Final | Dilution | Amt in 10 mL |
NaCl | 1.5 M | 0.5 M | 3 | 3.33 mL |
Tris-HCl | 500 mM | 20 mM | 25 | 400 uL |
EDTA | 0.5M | 1 mM | 500 | 20 uL |
nf H2O | NA | NA | NA | 6.25 mL |
Elution Buffer
Reagent | Stock | Final | Dilution | Amt in 1 mL (uL) |
Tris-HCl | 500 mM | 10 mM | 50 | 20 |
EDTA | 0.5M | 1 mM | 500 | 2 |
nf H2O | NA | NA | NA | 978 |
Low Salt Buffer
Reagent | Stock | Final | Dilution | Amt in 1 mL (uL) |
NaCl | 1.5 M | 0.15 M | 10 | 100 |
Tris-HCl | 500 mM | 20 mM | 25 | 40 |
EDTA | 0.5M | 1 mM | 500 | 2 |
nf H2O | NA | NA | NA | 858 |
Protocol
- Latch and Padlock Hybridization
- Mix together samples according to table; use 100 uL LPH buffer with 100 nM Padlock0001-68
- Incubate for 1 hr at 37C
- Wash twice with 100 uL wash buffer by pelleting cells at 600xg for 3 min
- Incubate cells for 20 min at 40C in 150 uL stringent wash buffer
- Resuspend pellet in 100 uL LPH buffer with 100 nM Latch0001
- For sample 3, use 10 uL 100 uM Latch0001 (10 uM final concentration)
- Incubate for 30 min at 37C
- Wash twice with 200 uL wash buffer by pelleting cells at 600xg for 3 min
- Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
- Resuspend with 20 uL 1x Amp Ligase buffer and transfer to a 0.2 mL tube
- Circularization
- Prepare 20 uL KLN mix
- Prepare 4.1x master mix of Phusion mix
- Add 20 uL Phusion mix to 20 uL of reaction for samples 2-5
- Add 2 uL KLN mix to 20 uL of reaction for sample 1
- Incubate at 55C for 4 hours
- Continued tomorrow
Sample No | Sample Name | LatchX2 | LatchX3 | PCAmp1 | PCAmp3 | Expected Product |
1 | Amp1 Test | X | X | Amp1 | ||
2 | Amp3 Test | X | X | Amp3 | ||
3 | LatchX2 Specificity | X | X | X | Amp1 | |
4 | LatchX3 Specificity | X | X | X | Amp3 | |
5 | No Template | X | X | None | ||
6 | No Latch | X | X | None | ||
7 | No Padlock | X | X | X | X | None |